1. ENERGY BALANCE JOURNAL CLUB
A High-Fat Diet Activates Oncogenic Kras and COX2 to Induce Development of
Pancreatic Ductal Adenocarcinoma in Mice
Bincy Philip, Christina L. Roland, Jaroslaw Daniluk, Yan Liu, Deyali Chatterjee,
Sobeyda B. Gomez, Baoan Ji, Haojie Huang, Huamin Wang, Jason B. Fleming,
Craig D. Logsdon, Zobeida Cruz-Monserrate
Zobeida Cruz-Monserrate Ph.D.
Instructor
Cancer Biology Department

2. Pancreatic Cancer: A Deadly Disease 2013 Estimated US Cancer Deaths
Men 306,920
Women 273,430
Lung & bronchus 28%
Prostate %
Colon & rectum 9%
Pancreas 6%
Liver & intrahepatic 5% bile duct
Leukemia 4%
Esophagus 4%
Urinary bladder 4%
Non-Hodgkin % lymphoma
Kidney 3%
26% Lung & bronchus
14% Breast
9% Colon & rectum
7% Pancreas
5% Ovary
4% Leukemia
3% Non-Hodgkin lymphoma
3% Uterine corpus
2% Liver & intrahepatic bile duct
2% Brain and other nervous system
Source: American Cancer Society, 2013

3. Prevention and Early Detection
Pancreatic Ductal Adenocarcinoma (PDAC) is Deadly and Difficult to Diagnose Early
Highest death to incidence ratio (0.99) of all cancers
5-year survival rate below 6%
Median survival ~ 6 months
Surgical resection is the only effective treatment
< 20% of the patients are eligible
Rarely detected at an early stage (small lesions)
High rate of dissemination
Conventional cancer treatments fail
Resistance to chemotherapy
Treatment options limited
Prevention and Early Detection

4. Pancreas Microanatomy
Endocrine
Exocrine
The pancreas is the organ responsible for regulating protein and carbohydrate
digestion as well as glucose homeostasis. It is regionally divided into the head, neck,
body and tail and is composed of two systems: the exocrine and endocrine pancreas.
The exocrine pancreas is arranged in a branching network of acinar and ductal cells that
produce digestive enzymes which are secreted into the duodenum. The endocrine pancreas
is composed of islet cells that produce hormones that are transported via the bloodstream
and regulate metabolism and glucose homeostasis.
Most pancreatic cancers (around 75%) develop specifi cally in the exocrine pancreas
Most pancreatic cancers (around 75%)
Develop in the exocrine pancreas
Omary, M.B., et. al., : p. 50-9

5. PDAC >90% Mutant K-Ras
Multistep Progression Model of PDAC
Pancreatic Intraepithelial Neoplasias (PanINs)
Normal duct
single cell layer
low cuboidal
PanIN-1A/1B
elongated cells
mucin
papillary growth
PanIN-2
early nuclear abnormalities
PanIN-3
luminal budding
nuclear atypia
mitosis
Carcinoma
invasion
desmoplasia
PDAC >90% Mutant K-Ras
Hruban, R.H., et al., (5): p

6. Non-Modifiable PDAC Risk Factors

7. Modifiable PDAC Risk Factors
Obesity

8. Obesity is a Risk Factor for Many Cancers Including Pancreatic
One of the major risk factors that has been linked to pancreatic cancer is obesity. Several cohort studies have stated that an increase in BMI is linked to an elevated risk of pancreatic cancer. Specifically, this paper pooled together 12 epidemiologic cohorts and one case control study and noticed that excess body fat has a direct linear relationship with increased risk of pancreatic cancer therefore supporting the fact that obesity can be a major risk factor.
Excess body weight is also an issue because it can be linked to diabetes which is another risk of pancreatic cancer. This works in that increasing fat results in an increase of insulin production to balance the glucose levels in the blood. This results in hyperinsulenmia. The more production of insulin, the more pancreatic cancer cells grow leading to the progression of cancer.
Eugenia E. Calle* and Rudolf Kaaks 2004:4:

9. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Cell Type Specific Promoter
Duct
Islet
Acini

10. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Cell Type Specific Promoter
Duct
Islet
Acini
Elastase-Cre-Er
Tamoxifen Regulated

11. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Cell Type Specific Promoter
Duct
Islet
Acini
Acinar Cell Specific Cre
Elastase-Cre-Er
Tamoxifen Regulated

12. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Oncogenic K-Ras
“flox-stopped“ X
Cell Type Specific Promoter
Endogenous Promoter “Knock In”
Stop
K-RasG12D
Poly A
p-K-Ras
Duct
loxP
loxP
Islet
loxP = locus of recombination
Acini
Acinar Cell Specific Cre
Elastase-Cre-Er
Tamoxifen Regulated

13. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Oncogenic K-Ras
“flox-stopped“ X
Cell Type Specific Promoter
Endogenous Promoter “Knock In”
Elastase-Cre-Er
Stop
K-RasG12D
Poly A
p-K-Ras
Cre mediated deletion via Tamoxifen after birth
loxP
loxP
loxP = locus of recombination
LSL/BAC
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells

14. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Oncogenic K-Ras
“flox-stopped“ X
Cell Type Specific Promoter
Endogenous Promoter “Knock In”
Elastase-Cre-Er
Stop
K-RasG12D
Poly A
p-K-Ras
Cre mediated deletion via Tamoxifen after birth
loxP
loxP
loxP = locus of recombination
LSL/BAC
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
Normal Pancreas at early age

15. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Oncogenic K-Ras
“flox-stopped“ X
Could this mouse model be used to test risk factors like obesity?
Cell Type Specific Promoter
Endogenous Promoter “Knock In”
Elastase-Cre-Er
Stop
K-RasG12D
Poly A
p-K-Ras
Cre mediated deletion via Tamoxifen after birth
loxP
loxP
loxP = locus of recombination
LSL/BAC
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
Normal Pancreas at early age

16. High Fat-Induced PDAC Mouse Model
LSL/BAC
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
We chose a isocaloric diets-solid low fat diet with 10% fat and a solid high fat diet with 61.6% fat for this experiment. We induced the mice with tamoxifen and started the mice on respective diets and fed the mice these diets for 30 days. On day 30 we sacrificed the mice and harvested the pancreas and collected the body and pancreas weight.
Purified diets on the other hand are created with purified ingredients like casein, corn starch, cellulose, and other vitamins and minerals. Each individual nutrient is provided by one individual ingredients so it gives greater control of the nutrient content of the diet. You can also remove nutrients easily so there is very little variation in each diet. So since we wanted to just study the effect of high fat, we could easily increase the level of fat in our diet.
These human food grade ingredients have relatively simple chemical compositions (predominantly one nutrient classification) and this feature is important for manipulating individual nutrients for research purposes. Additionally, most refined ingredients contain very limited levels of non-nutrients that could have biological activity. This is in contrast to the natural ingredients (corn, wheat, soybean meal, etc.) used in standard diets, which have relatively complex chemical compositions as well as various phytochemicals that may or may not be physiologically relevant.

17. High Fat-Induced PDAC Mouse Model
LSL/BAC
% kcal of each nutrient
Caloric
Breakdown
Control Diet
High Fat Diet
Protein
18.3
18.1
Fat
10.2
61.6
Carbohydrate
71.5
20.3
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
Isocaloric
We chose a isocaloric diets-solid low fat diet with 10% fat and a solid high fat diet with 61.6% fat for this experiment. We induced the mice with tamoxifen and started the mice on respective diets and fed the mice these diets for 30 days. On day 30 we sacrificed the mice and harvested the pancreas and collected the body and pancreas weight.
Purified diets on the other hand are created with purified ingredients like casein, corn starch, cellulose, and other vitamins and minerals. Each individual nutrient is provided by one individual ingredients so it gives greater control of the nutrient content of the diet. You can also remove nutrients easily so there is very little variation in each diet. So since we wanted to just study the effect of high fat, we could easily increase the level of fat in our diet.
These human food grade ingredients have relatively simple chemical compositions (predominantly one nutrient classification) and this feature is important for manipulating individual nutrients for research purposes. Additionally, most refined ingredients contain very limited levels of non-nutrients that could have biological activity. This is in contrast to the natural ingredients (corn, wheat, soybean meal, etc.) used in standard diets, which have relatively complex chemical compositions as well as various phytochemicals that may or may not be physiologically relevant.

18. High Fat-Induced PDAC Mouse Model
LSL/BAC
% kcal of each nutrient
Caloric
Breakdown
Control Diet
High Fat Diet
Protein
18.3
18.1
Fat
10.2
61.6
Carbohydrate
71.5
20.3
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
Isocaloric
We chose a isocaloric diets-solid low fat diet with 10% fat and a solid high fat diet with 61.6% fat for this experiment. We induced the mice with tamoxifen and started the mice on respective diets and fed the mice these diets for 30 days. On day 30 we sacrificed the mice and harvested the pancreas and collected the body and pancreas weight.
Purified diets on the other hand are created with purified ingredients like casein, corn starch, cellulose, and other vitamins and minerals. Each individual nutrient is provided by one individual ingredients so it gives greater control of the nutrient content of the diet. You can also remove nutrients easily so there is very little variation in each diet. So since we wanted to just study the effect of high fat, we could easily increase the level of fat in our diet.
These human food grade ingredients have relatively simple chemical compositions (predominantly one nutrient classification) and this feature is important for manipulating individual nutrients for research purposes. Additionally, most refined ingredients contain very limited levels of non-nutrients that could have biological activity. This is in contrast to the natural ingredients (corn, wheat, soybean meal, etc.) used in standard diets, which have relatively complex chemical compositions as well as various phytochemicals that may or may not be physiologically relevant.
CONTROL DIET
HIGH FAT DIET
BAC
(n=10)
LSL/BAC

19. High Fat Diet Increased Total Body Weight and Pancreas Weight Compared to Control Diet
I had weighed the mice every week that they were on the respective diets to monitor if high fat diet was causing the mice to become obese. I graphed the percent change in body weight from day 1 to day 30. Animals on high fat diet had a larger change in body weight compared to the animals on control diet as we had expected showing that the diet was indeed making the mice obese. I also graphed the total pancreas weight over the total body weight to see if the pancreas of mice on high fat were fatter than the animals on the other diets. LSL/BAC mice on high fat diet had a higher pancreas weight compared to control diet LSL/BAC mice.

20. High Fat Diet Increased Inflammation, Fibrosis, and PanIN Lesions on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

21. High Fat Diet Increased Inflammation, Fibrosis, and PanIN Lesions on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

22. High Fat Diet Increased Areas of Collagen Deposition on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

23. High Fat Diet Increased Areas of Activated Stellate Cells on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.
Stellate Cells

24. Endogenous Mutant K-Ras is not Sufficient to Transform Most Cells
Developmental promoter EMBRYONIC expression in all cell types
Multiple PanIN lesions; PDAC- 2/29 (7%) 1 year
Endogenous K-Ras mutations generates cancer with low efficiency
Acinar cell promoter
ADULT expression in acinar cells
No PanIns No tumors – 0/11 (0%) 1 year
(Unless inflammation was induced)

25. 100% specific for adult acinar cells
Mutant K-Ras at Endogenous Levels Requires an Inflammatory Insult to Transform Acinar Cells
Bac-Elastase CreERT
100% efficient
100% specific for adult acinar cells
If oncogenic mutant Ras is always “on” then there should be effects on cell function.

26. Mutant K-Ras at Endogenous Levels Requires an Inflammatory Insult to Transform Acinar Cells
mutant Ras NO Stimulant
mutant Ras
+ LPS Stimulant

27. Link Between Ras and Obesity?
High Fat Diet
Link Between Ras and Obesity?

28. High Fat Diet Increased Ras Activity in Mice with K-RasG12D Mutation
I specifically wanted to determine if the high fat diet was in fact aiding in activation of Ras and if it had the ability to increase its activity. For this reason I performed a Raf pulldown assay which utilizes a Ras-binding domain (RBD) of the Ras effector kinase Raf1.  The Raf-RBD domain has been shown to bind specifically to the GTP-bound form of Ras proteins. I performed this assay several times and I pooled the results and I calculated the fold change compared to low fat BAC. The level of active Ras in LSL/BAC animals on high fat diet was elevated compared to LSL/BAC on control diet and the BAC controls on both diets.

29. High Fat Diet Activates of K-Ras Downstream Pathways in Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

30. Ras Must Be Active to Initiate Downstream Signaling Resulting in Progression of PDAC
Cox2
PGE2
Receptors (GF, hormones, etc)
PI3K
Ras-GTP
GEFs
Ras-GDP
Raf
GAPs
RalGDS
INACTIVE
ACTIVE
Ras is a protein that is present normally in our bodies and functions to turn on signaling pathways that regulate cell growth and differentiation. In a normal cell Ras is bound to GDP and is in its inactive state. Receptors such as growth factors and hormones activate GEFs which are guanine exchange factors remove GDP and bind GTP to Ras. When Ras is GTP bound, it is active and able to turn on downstream signaling pathways like MAPK and PI3K and Cox-2 which are important for cell growth, proliferation, differentiation, etc. In a normal cell, due to GTP hydrolysis and the activation of GAPs (GTPase activating protein), the GTP is removed and GDP is bound to Ras bringing it back to its inactive state. So in a normal cell the function of Ras is very transient. However, when an oncogenic mutation is present in Ras, it blocks the GAP interaction and reduces the GTPase activity so Ras is constitutively active. However, according to data from our lab, when mutant K-ras is expressed as in the Tyler Jacks K-ras model, the mutant K-ras is bound to GDP so its in an inactive state. Something like an inflammatory stimulus or another protein needs to activate GEFs and turn on Ras for it to be constitutively active so oncogenic Ras is not ON till it is turned on. Now once mutant K-ras is activated, it turns on downstream targets like Cox-2 which activates the expression of PGE2 which are normally involved in modulation of immune responses, protection of the gastrointestinal mucosa, promote inflammation, swelling, pain and fever. PGE2 activation by Cox-2 drives this feed forward loops thus increasing the activity of Ras. Therefore, oncogenic Ras activity is prolonged.
MAPK

31. High Fat Diet Increases Cox-2 in Pancreas on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

32. High Fat Diet Promotes Recruitment of Macrophages on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

33. High Fat Diet Increases Cox-2 in Pancreas on Mice with K-RasG12D Mutation
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

34. High Fat Diet Promotes PDAC
LSL/BAC
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
Normal Pancreas at early age

35. High Fat Diet Promotes PDAC
LSL/BAC
Acinar
mK-Ras
High Fat Diet
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
Normal Pancreas at early age
Cox2
PanINs
Cancer

36. Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras
LSL/BAC
Cox-2 KO
“flox-stopped“
Acinar
mK-Ras X
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells

37. Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras
LSL/BAC
Cox-2 KO
“flox-stopped“
Acinar
mK-Ras X
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells
COXKO/LSL/BAC
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells but NO Cox-2 Expression

38. COX-2 is Required in High Fat-Induced PDAC Mouse Model
COXKO/LSL/BAC
% kcal of each nutrient
Caloric
Breakdown
Control Diet
High Fat Diet
Protein
18.3
18.1
Fat
10.2
61.6
Carbohydrate
71.5
20.3
Acinar
mK-Ras
K-RasG12D
Endogenous
mutant K-Ras expression in acinar cells but NO Cox-2 Expression
Isocaloric
We chose a isocaloric diets-solid low fat diet with 10% fat and a solid high fat diet with 61.6% fat for this experiment. We induced the mice with tamoxifen and started the mice on respective diets and fed the mice these diets for 30 days. On day 30 we sacrificed the mice and harvested the pancreas and collected the body and pancreas weight.
Purified diets on the other hand are created with purified ingredients like casein, corn starch, cellulose, and other vitamins and minerals. Each individual nutrient is provided by one individual ingredients so it gives greater control of the nutrient content of the diet. You can also remove nutrients easily so there is very little variation in each diet. So since we wanted to just study the effect of high fat, we could easily increase the level of fat in our diet.
These human food grade ingredients have relatively simple chemical compositions (predominantly one nutrient classification) and this feature is important for manipulating individual nutrients for research purposes. Additionally, most refined ingredients contain very limited levels of non-nutrients that could have biological activity. This is in contrast to the natural ingredients (corn, wheat, soybean meal, etc.) used in standard diets, which have relatively complex chemical compositions as well as various phytochemicals that may or may not be physiologically relevant.
CONTROL DIET
HIGH FAT DIET
COXKO/BAC
(n=10)
COXKO/LSL/BAC

39. Conditional Knockout of Cox-2 in the Acinar Cells Blocked the Effects of High Fat Diet
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

40. Conditional Knockout of Cox-2 in the Acinar Cells Blocked the Effects of High Fat Diet
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

41. Conditional Knockout of Cox-2 in the Acinar Cells Blocked the Effects of High Fat Diet
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

42. Systemic Cox-2 inhibition Decreases the Effects of High Fat Diet
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

43. Systemic Cox-2 inhibition Decreases the Effects of High Fat Diet
When we looked at the pancreases of these animals, we saw a drastic difference between groups. Animals lacking oncogenic K-ras expression or the BAC littermate control animals were healthy when they were fed either a control diet and a high fat diet. However, if you look at the LSL/BAC animals that express mutant K-ras, animals treated with a high fat diet had larger areas of fibrosis with PanIN formation while the animals on control diet had small areas of fibrosis. So the histology itself shows clearly that consumption of a high fat diet in the presence of mutant K-ras initiates the early stages of cancer development faster than animals on control diet.

44. High Fat Diet Decreases Survival of Mice Susceptible to PDAC
30 days

45. High Fat Diet Decreases Survival of Mice Susceptible to PDAC
Control Diet
30 days
160
Days
High Fat Diet

46. High Fat Diet Decreases Survival of Mice Susceptible to PDAC
Control Diet
High Fat Diet
30 days
205
Days
Pancreas
Pancreas
Pancreas

47. High Fat Diet Decreases Survival of Mice Susceptible to PDAC
Control Diet
High Fat Diet
30 days
205
Days
Pancreas
Pancreas
High Fat Diet
Pancreas
Pancreas

48. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Cell Type Specific Promoter
Duct
Islet
Acini
Elastase-Cre-Er
Tamoxifen Regulated

49. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Cell Type Specific Promoter
PDX-1 Cre
Activates Cre during development

50. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Oncogenic K-Ras
“flox-stopped“ X
Cell Type Specific Promoter
Endogenous Promoter “Knock In”
PDX-1 Cre
Stop
K-RasG12D
Poly A
Activates Cre during development
p-K-Ras
loxP
loxP
loxP = locus of recombination

51. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Rasre
Oncogenic K-Ras
“flox-stopped“ X
Cell Type Specific Promoter
Endogenous Promoter “Knock In”
PDX-1 Cre
Stop
K-RasG12D
Poly A
Activates Cre during embryogenesis
p-K-Ras
loxP
loxP
loxP = locus of recombination
Cre mediated deletion during development
LSL/PDX-1
mK-Ras
All cells
K-RasG12D
Endogenous
mutant K-Ras expression in every cell of the pancreas

52. High Fat Diet Decreases Survival of Mice Susceptible to PDAC
Some
Mice in control diet are still alive
Cre mediated deletion via Tamoxifen after birth
Cre mediated deletion during development

53. Cre mediated deletion during development
High Fat Diet Accelerated PanIN-2 and PanIN-3 Development Compared to Control Diet Using and Embryonic Promoter
Control Diet
137
Days
High Fat Diet
Cre mediated deletion during development 90
Days

54. Cre mediated deletion during development
High Fat Diet Accelerated PanIN-2 and PanIN-3 Development Compared to Control Diet Using and Embryonic Promoter
Control Diet
137
Days
High Fat Diet
Cre mediated deletion during development 90
Days

56. Summary: Obesity is a Risk Factor for PDAC in Humans
High Fat Diet Accelerates PanIN formation and
Cancer Development in PDAC Mouse Models
Ras Activity and Cox-2 are essential
for High Fat Diet induced PDAC
HOW DOES FAT LEAD TO RAS ACTIVATION?
Mechanisms?

57. Future Directions with High Fat Induced PDAC Model
Test Prevention Agents
LSL/BAC

58. NIDDK Minority Supplement
Acknowledgments
Dr. Logsdon’s Laboratory
Collaborators
MDACC
Huamin Wang, Pathology
Jason Fleming, Surgical Oncology
Joya Chandra, Pediatrics
David McConkey, Urology
Kathleen M. Schmeler MD, Ob/GYN
Ralph B. Arlinghaus, Translational Molecular Pathology
Adel El-Naggar, Pathology
The Methodist Hospital Research Institute
Ching-Hsuan Tung Ph.D.
Wael R. Abd-Elgaliel Ph.D.
Rita Serta Ph.D.
City of Hope National Medical Center
Ann David Ph.D.
University of Rhode Island
Oleg Andreev
Yana Reshetnyak
Bincy Philips, MS
Funding Sources
NIDDK Minority Supplement
Phi Beta Psi Sorority